Snake venoms are mixtures of proteins and peptides that possess lethal properties in the context of snakebite envenomation but in small doses, translate well for promising therapeutics and biotechnological applications. To harness the potential of venom proteins, characterisation of venom proteomes and the structural heterogeneity therein is crucial. While technological advances of bottom-up proteomics have rapidly enabled profiling of snake venom proteomes at the toxin family level, the subtle structural heterogeneity present in unique protein isoforms (proteoforms) are difficult to capture with conventional proteomic methods alone. This underpins the need for complementary bioanalytical tools to aid venom heterogeneity characterisation.
Top-down proteomics is a promising complementary technique for protein sequencing without the need for prior enzymatic digestion, thus capturing crucial proteoform information at the intact level and has been used to interrogate proteoforms across different fields. Here, we describe the integrated use of top-down proteomics and other mass spectrometry-based techniques to explore venom proteoforms from the forest cobra (Naja melanoleuca) with an emphasis on the unique technological capabilities of cyclic ion mobility mass spectrometry. We explored different gas-phase strategies that could improve identification on select venom proteoforms. Of note, we compared a hybrid fragmentation approach to a conventional collision induced dissociation method and demonstrated protein sequence coverage could be bolstered from 80 % to 97 % for a phospholipase A2 proteoform. Furthermore, we showcased the unique instrumentation of the cyclic ion mobility cell which enabled custom multifunctional separation experiments in the gas-phase, improving identification of the phospholipase A2 proteoform to full sequence coverage by filtering spectral noise and enhancing protein signal. This workflow highlights promising technological additions to existing top-down proteomic strategies and complements traditional bottom-up proteomic methods. We envisage its far-reaching applications to go beyond venoms and be of service to other proteoforms across different biological systems.